Full-Genome Sequence of Human Betacoronavirus 2c Jordan-N3/2012 after Serial Passage in Mammalian Cells
Identifieur interne : 000693 ( Pmc/Corpus ); précédent : 000692; suivant : 000694Full-Genome Sequence of Human Betacoronavirus 2c Jordan-N3/2012 after Serial Passage in Mammalian Cells
Auteurs : Kenneth G. Frey ; Cassie L. Redden ; Kimberly A. Bishop-Lilly ; Reed Johnson ; Lisa E. Hensley ; Kanakatte Raviprakash ; Thomas Luke ; Tad Kochel ; Vishwesh P. Mokashi ; Gabriel N. DefangSource :
- Genome Announcements [ 2169-8287 ] ; 2014.
Abstract
Middle East respiratory syndrome coronavirus (MERS-CoV) is the etiologic agent of a highly lethal pneumonia. Here, we report the full-genome sequence of the Jordan-N3/2012 strain after serial passage in two distinct mammalian cell lines. The genome exhibits noteworthy stability, which may inform the development of vaccines and therapeutics used to treat infection with this virus.
Url:
DOI: 10.1128/genomeA.00324-14
PubMed: 24874668
PubMed Central: 4038873
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<record><TEI><teiHeader><fileDesc><titleStmt><title xml:lang="en">Full-Genome Sequence of Human Betacoronavirus 2c Jordan-N3/2012 after Serial Passage in Mammalian Cells</title><author><name sortKey="Frey, Kenneth G" sort="Frey, Kenneth G" uniqKey="Frey K" first="Kenneth G." last="Frey">Kenneth G. Frey</name></author><author><name sortKey="Redden, Cassie L" sort="Redden, Cassie L" uniqKey="Redden C" first="Cassie L." last="Redden">Cassie L. Redden</name></author><author><name sortKey="Bishop Lilly, Kimberly A" sort="Bishop Lilly, Kimberly A" uniqKey="Bishop Lilly K" first="Kimberly A." last="Bishop-Lilly">Kimberly A. Bishop-Lilly</name></author><author><name sortKey="Johnson, Reed" sort="Johnson, Reed" uniqKey="Johnson R" first="Reed" last="Johnson">Reed Johnson</name><affiliation><nlm:aff id="aff4">National Institutes of Health, National Institute of Allergy and Infectious Diseases, Integrated Research Facility, Fort Detrick, Maryland, USA</nlm:aff></affiliation></author><author><name sortKey="Hensley, Lisa E" sort="Hensley, Lisa E" uniqKey="Hensley L" first="Lisa E." last="Hensley">Lisa E. Hensley</name><affiliation><nlm:aff id="aff4">National Institutes of Health, National Institute of Allergy and Infectious Diseases, Integrated Research Facility, Fort Detrick, Maryland, USA</nlm:aff></affiliation></author><author><name sortKey="Raviprakash, Kanakatte" sort="Raviprakash, Kanakatte" uniqKey="Raviprakash K" first="Kanakatte" last="Raviprakash">Kanakatte Raviprakash</name><affiliation><nlm:aff id="aff3">Naval Medical Research Center, Viral and Rickettsial Diseases Department, Silver Spring, Maryland, USA</nlm:aff></affiliation></author><author><name sortKey="Luke, Thomas" sort="Luke, Thomas" uniqKey="Luke T" first="Thomas" last="Luke">Thomas Luke</name></author><author><name sortKey="Kochel, Tad" sort="Kochel, Tad" uniqKey="Kochel T" first="Tad" last="Kochel">Tad Kochel</name><affiliation><nlm:aff id="aff3">Naval Medical Research Center, Viral and Rickettsial Diseases Department, Silver Spring, Maryland, USA</nlm:aff></affiliation></author><author><name sortKey="Mokashi, Vishwesh P" sort="Mokashi, Vishwesh P" uniqKey="Mokashi V" first="Vishwesh P." last="Mokashi">Vishwesh P. Mokashi</name><affiliation><nlm:aff id="aff1">Naval Medical Research Center, NMRC-Frederick, Fort Detrick, Maryland, USA</nlm:aff></affiliation></author><author><name sortKey="Defang, Gabriel N" sort="Defang, Gabriel N" uniqKey="Defang G" first="Gabriel N." last="Defang">Gabriel N. Defang</name><affiliation><nlm:aff id="aff5">Viral and Zoonotic Diseases Research Program, U.S. Naval Medical Research Unit 3, Abbassia, Cairo, Egypt</nlm:aff></affiliation></author></titleStmt><publicationStmt><idno type="wicri:source">PMC</idno><idno type="pmid">24874668</idno><idno type="pmc">4038873</idno><idno type="url">http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4038873</idno><idno type="RBID">PMC:4038873</idno><idno type="doi">10.1128/genomeA.00324-14</idno><date when="2014">2014</date><idno type="wicri:Area/Pmc/Corpus">000693</idno><idno type="wicri:explorRef" wicri:stream="Pmc" wicri:step="Corpus" wicri:corpus="PMC">000693</idno></publicationStmt><sourceDesc><biblStruct><analytic><title xml:lang="en" level="a" type="main">Full-Genome Sequence of Human Betacoronavirus 2c Jordan-N3/2012 after Serial Passage in Mammalian Cells</title><author><name sortKey="Frey, Kenneth G" sort="Frey, Kenneth G" uniqKey="Frey K" first="Kenneth G." last="Frey">Kenneth G. Frey</name></author><author><name sortKey="Redden, Cassie L" sort="Redden, Cassie L" uniqKey="Redden C" first="Cassie L." last="Redden">Cassie L. Redden</name></author><author><name sortKey="Bishop Lilly, Kimberly A" sort="Bishop Lilly, Kimberly A" uniqKey="Bishop Lilly K" first="Kimberly A." last="Bishop-Lilly">Kimberly A. Bishop-Lilly</name></author><author><name sortKey="Johnson, Reed" sort="Johnson, Reed" uniqKey="Johnson R" first="Reed" last="Johnson">Reed Johnson</name><affiliation><nlm:aff id="aff4">National Institutes of Health, National Institute of Allergy and Infectious Diseases, Integrated Research Facility, Fort Detrick, Maryland, USA</nlm:aff></affiliation></author><author><name sortKey="Hensley, Lisa E" sort="Hensley, Lisa E" uniqKey="Hensley L" first="Lisa E." last="Hensley">Lisa E. Hensley</name><affiliation><nlm:aff id="aff4">National Institutes of Health, National Institute of Allergy and Infectious Diseases, Integrated Research Facility, Fort Detrick, Maryland, USA</nlm:aff></affiliation></author><author><name sortKey="Raviprakash, Kanakatte" sort="Raviprakash, Kanakatte" uniqKey="Raviprakash K" first="Kanakatte" last="Raviprakash">Kanakatte Raviprakash</name><affiliation><nlm:aff id="aff3">Naval Medical Research Center, Viral and Rickettsial Diseases Department, Silver Spring, Maryland, USA</nlm:aff></affiliation></author><author><name sortKey="Luke, Thomas" sort="Luke, Thomas" uniqKey="Luke T" first="Thomas" last="Luke">Thomas Luke</name></author><author><name sortKey="Kochel, Tad" sort="Kochel, Tad" uniqKey="Kochel T" first="Tad" last="Kochel">Tad Kochel</name><affiliation><nlm:aff id="aff3">Naval Medical Research Center, Viral and Rickettsial Diseases Department, Silver Spring, Maryland, USA</nlm:aff></affiliation></author><author><name sortKey="Mokashi, Vishwesh P" sort="Mokashi, Vishwesh P" uniqKey="Mokashi V" first="Vishwesh P." last="Mokashi">Vishwesh P. Mokashi</name><affiliation><nlm:aff id="aff1">Naval Medical Research Center, NMRC-Frederick, Fort Detrick, Maryland, USA</nlm:aff></affiliation></author><author><name sortKey="Defang, Gabriel N" sort="Defang, Gabriel N" uniqKey="Defang G" first="Gabriel N." last="Defang">Gabriel N. Defang</name><affiliation><nlm:aff id="aff5">Viral and Zoonotic Diseases Research Program, U.S. Naval Medical Research Unit 3, Abbassia, Cairo, Egypt</nlm:aff></affiliation></author></analytic><series><title level="j">Genome Announcements</title><idno type="eISSN">2169-8287</idno><imprint><date when="2014">2014</date></imprint></series></biblStruct></sourceDesc></fileDesc><profileDesc><textClass></textClass></profileDesc></teiHeader><front><div type="abstract" xml:lang="en"><p>Middle East respiratory syndrome coronavirus (MERS-CoV) is the etiologic agent of a highly lethal pneumonia. Here, we report the full-genome sequence of the Jordan-N3/2012 strain after serial passage in two distinct mammalian cell lines. The genome exhibits noteworthy stability, which may inform the development of vaccines and therapeutics used to treat infection with this virus.</p></div></front><back><div1 type="bibliography"><listBibl><biblStruct><analytic><author><name sortKey="Zaki, Am" uniqKey="Zaki A">AM Zaki</name></author><author><name sortKey="Van Boheemen, S" uniqKey="Van Boheemen S">S van Boheemen</name></author><author><name sortKey="Bestebroer, Tm" uniqKey="Bestebroer T">TM Bestebroer</name></author><author><name sortKey="Osterhaus, Ad" uniqKey="Osterhaus A">AD Osterhaus</name></author><author><name sortKey="Fouchier, Ra" uniqKey="Fouchier R">RA Fouchier</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Van Boheemen, S" uniqKey="Van Boheemen S">S van Boheemen</name></author><author><name sortKey="De Graaf, M" uniqKey="De Graaf M">M de Graaf</name></author><author><name sortKey="Lauber, C" uniqKey="Lauber C">C Lauber</name></author><author><name sortKey="Bestebroer, Tm" 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<front><journal-meta><journal-id journal-id-type="nlm-ta">Genome Announc</journal-id><journal-id journal-id-type="iso-abbrev">Genome Announc</journal-id><journal-id journal-id-type="hwp">ga</journal-id><journal-id journal-id-type="pmc">ga</journal-id><journal-id journal-id-type="publisher-id">GA</journal-id><journal-title-group><journal-title>Genome Announcements</journal-title></journal-title-group><issn pub-type="epub">2169-8287</issn><publisher><publisher-name>American Society for Microbiology</publisher-name><publisher-loc>1752 N St., N.W., Washington, DC</publisher-loc></publisher></journal-meta><article-meta><article-id pub-id-type="pmid">24874668</article-id><article-id pub-id-type="pmc">4038873</article-id><article-id pub-id-type="publisher-id">genomeA00324-14</article-id><article-id pub-id-type="doi">10.1128/genomeA.00324-14</article-id><article-categories><subj-group subj-group-type="heading"><subject>Viruses</subject></subj-group></article-categories><title-group><article-title>Full-Genome Sequence of Human Betacoronavirus 2c Jordan-N3/2012 after Serial Passage in Mammalian Cells</article-title></title-group><contrib-group><contrib contrib-type="author" corresp="yes"><name><surname>Frey</surname><given-names>Kenneth G.</given-names></name><xref ref-type="aff" rid="aff1 aff2"><sup>a,b</sup></xref></contrib><contrib contrib-type="author"><name><surname>Redden</surname><given-names>Cassie L.</given-names></name><xref ref-type="aff" rid="aff1 aff2"><sup>a,b</sup></xref></contrib><contrib contrib-type="author"><name><surname>Bishop-Lilly</surname><given-names>Kimberly A.</given-names></name><xref ref-type="aff" rid="aff1 aff2"><sup>a,b</sup></xref></contrib><contrib contrib-type="author"><name><surname>Johnson</surname><given-names>Reed</given-names></name><xref ref-type="aff" rid="aff4"><sup>d</sup></xref></contrib><contrib contrib-type="author"><name><surname>Hensley</surname><given-names>Lisa E.</given-names></name><xref ref-type="aff" rid="aff4"><sup>d</sup></xref></contrib><contrib contrib-type="author"><name><surname>Raviprakash</surname><given-names>Kanakatte</given-names></name><xref ref-type="aff" rid="aff3"><sup>c</sup></xref></contrib><contrib contrib-type="author"><name><surname>Luke</surname><given-names>Thomas</given-names></name><xref ref-type="aff" rid="aff2 aff3"><sup>b,c</sup></xref></contrib><contrib contrib-type="author"><name><surname>Kochel</surname><given-names>Tad</given-names></name><xref ref-type="aff" rid="aff3"><sup>c</sup></xref></contrib><contrib contrib-type="author"><name><surname>Mokashi</surname><given-names>Vishwesh P.</given-names></name><xref ref-type="aff" rid="aff1"><sup>a</sup></xref></contrib><contrib contrib-type="author" corresp="yes"><name><surname>Defang</surname><given-names>Gabriel N.</given-names></name><xref ref-type="aff" rid="aff5"><sup>e</sup></xref></contrib><aff id="aff1"><label>a</label>Naval Medical Research Center, NMRC-Frederick, Fort Detrick, Maryland, USA</aff><aff id="aff2"><label>b</label>Henry M. Jackson Foundation, Bethesda, Maryland, USA</aff><aff id="aff3"><label>c</label>Naval Medical Research Center, Viral and Rickettsial Diseases Department, Silver Spring, Maryland, USA</aff><aff id="aff4"><label>d</label>National Institutes of Health, National Institute of Allergy and Infectious Diseases, Integrated Research Facility, Fort Detrick, Maryland, USA</aff><aff id="aff5"><label>e</label>Viral and Zoonotic Diseases Research Program, U.S. Naval Medical Research Unit 3, Abbassia, Cairo, Egypt</aff></contrib-group><author-notes><corresp id="cor1">Address correspondence to Kenneth G. Frey, <email>kenneth.frey@med.navy.mil</email>, or Gabriel N. Defang, <email>gabriel.defang@med.navy.mil</email>.</corresp></author-notes><pub-date pub-type="epub"><day>29</day><month>5</month><year>2014</year></pub-date><pub-date pub-type="collection"><season>May-Jun</season><year>2014</year></pub-date><volume>2</volume><issue>3</issue><elocation-id>e00324-14</elocation-id><history><date date-type="received"><day>27</day><month>3</month><year>2014</year></date><date date-type="accepted"><day>13</day><month>5</month><year>2014</year></date></history><permissions><copyright-statement>Copyright © 2014 Frey et al.</copyright-statement><copyright-year>2014</copyright-year><copyright-holder>Frey et al.</copyright-holder><license license-type="open-access" xlink:href="http://creativecommons.org/licenses/by/3.0/"><license-p>This is an open-access article distributed under the terms of the <ext-link ext-link-type="uri" xlink:href="http://creativecommons.org/licenses/by/3.0/">Creative Commons Attribution 3.0 Unported license</ext-link>.</license-p></license></permissions><self-uri xlink:title="pdf" xlink:type="simple" xlink:href="jga003141323001.pdf"></self-uri><abstract><p>Middle East respiratory syndrome coronavirus (MERS-CoV) is the etiologic agent of a highly lethal pneumonia. Here, we report the full-genome sequence of the Jordan-N3/2012 strain after serial passage in two distinct mammalian cell lines. The genome exhibits noteworthy stability, which may inform the development of vaccines and therapeutics used to treat infection with this virus.</p></abstract><counts><page-count count="2"></page-count></counts><custom-meta-group><custom-meta><meta-name>cover-date</meta-name><meta-value>May/June 2014</meta-value></custom-meta><custom-meta><meta-name>access-type</meta-name><meta-value>free</meta-value></custom-meta></custom-meta-group></article-meta></front><body><sec><title>GENOME ANNOUNCEMENT</title><p>In October 2012, an unknown betacoronavirus was isolated from a Saudi man with acute pneumonia (<xref rid="B1" ref-type="bibr">1</xref>), and the genome sequence of this virus was announced shortly thereafter (<xref rid="B2" ref-type="bibr">2</xref>). Reports followed of a similar virus isolated from patients in Qatar, England, and Jordan (<xref rid="B3" ref-type="bibr">3</xref>, <xref rid="B4" ref-type="bibr">4</xref>). Subsequently, the virus was designated Middle East respiratory syndrome coronavirus (MERS-CoV) (<xref rid="B5" ref-type="bibr">5</xref>). A recent WHO report identified 180 laboratory-confirmed cases of MERS-CoV, including 77 deaths (<xref rid="B6" ref-type="bibr">6</xref>). Numerous investigations are being conducted, including efforts to develop vaccines and therapeutics.</p><p>It has been reported that MERS-CoV is capable of replicating in a wide variety of mammalian cell types (<xref rid="B7" ref-type="bibr">7</xref>). However, it was unclear if serial growth impacts the viral genome. Here, we report the whole-genome sequence of MERS-CoV Jordan-N3/2012 after sequential passage in Vero CCL81 cells and a human embryonic fibroblast line (MRC5). The genome of MERS-CoV is a single-stranded RNA (ssRNA) encoding 10 proteins: a replicase polyprotein, (ORF1ab), three structural proteins (E, N, and M), a surface glycoprotein (S), and five nonstructural proteins (open reading frame [ORF] 3, 4a, 4b, 5, and 8b) (<xref rid="B2" ref-type="bibr">2</xref>).</p><p>Total RNA was extracted from the supernatants of infected cell cultures, and sequencing libraries were created using the TruSeq RNA sample prep version 2 kit (Illumina, Inc.), beginning the protocol at the fragmentation step. Paired-end 151-base sequencing was performed on the MiSeq (average >730 Mb/sample). The reads were aligned against the NCBI reference (accession no. KC776147.1) using the CLC bio Genomics Workbench (version 6.5). In addition, the sequence reads were <italic>de novo</italic> assembled using CLC bio.</p><p>In total, five samples were sequenced: passages 2, 6, 7, and 8 through CCL81 cells and passage 2 through MRC5 cells. All samples shared two single-nucleotide variants (SNVs) compared to the reference strain Jordan-N3/2012. Further analysis of putative SNVs indicated that all samples share an SNV at reference position 24045. This T→C transversion falls in the S gene and results in a nonsynonymous mutation, I→T. This variant is present in 55.8% of the reads in the MRC5 passage and 73.4% of the reads in the CCL81 passage. The proportion of reads with this SNV increased in the Vero-passaged samples, from 34.55% in passage 6 to 93.89% in passage 8, likely representing a cell culture adaptation. This residue is not predicted to reside in the binding domain of the S protein (<xref rid="B8" ref-type="bibr">8</xref>). Although the reference genome contains an ambiguous nucleotide (W) at position 11262, all samples show a distinct preference for T (resulting in a leucine versus a histidine residue). It is uncertain as to whether this variation is due to a sequencing error in the reference genome or if population-level differences were lost during cell passage. Although this SNV is present in the coding sequence (CDS) of the ORF1ab gene, the functional consequences are unclear. The residue corresponding to this codon lies between the endopeptidase and the replicase domains and may not be present in mature virions. The apparent stability of this virus <italic>in vitro</italic> may facilitate the development of countermeasures by reducing the potential for rapid evolution and the resulting changes in immunodominant epitopes.</p><sec id="h1"><title>Nucleotide sequence accession number. </title><p>The genome sequence was deposited in GenBank under the accession no. <ext-link ext-link-type="uri" xlink:href="http://www.ncbi.nlm.nih.gov/nuccore?term=KJ614529">KJ614529</ext-link><bold>.</bold></p></sec></sec></body><back><fn-group><fn fn-type="other"><p><bold>Citation</bold> Frey KG, Redden CL, Bishop-Lilly KA, Johnson R, Hensley LE, Raviprakash K, Luke T, Kochel T, Mokashi VP, Defang GN. 2014. Full-genome sequence of human betacoronavirus 2c Jordan-N3/2012 after serial passage in mammalian cells. Genome Announc. 2(3):e00324-14. doi:10.1128/genomeA.00324-14.</p></fn></fn-group><ack><title>ACKNOWLEDGMENTS</title><p>The views expressed in this article are those of the authors and do not necessarily reflect the official policy or position of the Department of the Navy, Department of Defense, or the US. 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